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INFECTION AND IMMUNITY,0019-9567/00/$04.0010

Sept. 2000, p. 4972–4979 Vol. 68, No. 9

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Molecular Cloning, Sequencing, Expression, and Characterization ofan Immunogenic 43-Kilodalton Lipoprotein of Bartonella

bacilliformis That Has Homology to NlpD/LppBINDIRA PADMALAYAM,1,2* TIMOTHY KELLY,2 BARBARA BAUMSTARK,2

AND ROBERT MASSUNG1

Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333,1

and Department of Biology, Georgia State University, Atlanta, Georgia 303032

Received 17 February 2000/Returned for modification 24 March 2000/Accepted 28 May 2000

A recombinant clone expressing an immunoreactive antigen of Bartonella bacilliformis was isolated by screen-ing a genomic DNA library with serum from a patient with the chronic verruga phase of bartonellosis. Theclone, pBIPIM-17, contained a partial open reading frame that expressed an immunoreactive fusion protein.Subsequent rescreening of the library by plaque hybridization resulted in the isolation of recombinant clonesthat contain the entire open reading frame. The open reading frame (ORF-401) is capable of encoding a proteinof 401 amino acids with a predicted molecular mass of 43 kDa. The deduced amino acid sequence of theencoded protein was found to be highly homologous to a recently identified bacterial lipoprotein (LppB/NlpD)which has been associated with virulence. Evidence has been provided to show that the 43-kDa antigen ofB. bacilliformis is a lipoprotein and that it is likely to use the same biosynthetic pathway as other bacteriallipoproteins. This is the first report to date that characterizes a lipoprotein of B. bacilliformis. The immuno-genicity of the B. bacilliformis LppB homologue was demonstrated by Western blot analysis using sera frompatients with clinical bartonellosis. Sera from patients who had a high titer for Bartonella henselae, thecausative agent of bacillary angiomatosis and cat scratch disease, also recognized the recombinant 43-kDaantigen, suggesting that a homologue of this antigen is present in B. henselae. Using a cocktail of syntheticpeptides corresponding to predicted major antigenic sites, polyclonal antiserum specific for the LppB homo-logue of B. bacilliformis was generated. This antiserum did not recognize the NlpD homologue of Escherichia colior the 43-kDa antigen of B. henselae.

Bartonella bacilliformis is the etiologic agent of bartonellosis(Carrion’s disease), a unique biphasic disease that is prevalentamong inhabitants of the western slopes of the Andes Moun-tains in Columbia, Ecuador, and Peru. The primary phase ofthe disease is known as Oroya fever and is characterized by avery severe hemolytic anemia that was fatal in approximately40% of cases in the preantibiotic era. The cause of death isprimarily the severe anemia, in which nearly 100% of theerythrocytes are parasitized by bartonellae. Bartonellosis alsoinduces transient immunosuppression that results in the onsetof potentially life-threatening opportunistic infections suchas salmonellosis, shigellosis, and tuberculosis. The second-ary phase of bartonellosis, known as verruga peruana, mani-fests itself 4 to 8 weeks after the onset of Oroya fever. Thisphase is rarely fatal and is characterized by nodular eruptionsinvolving the face, neck, and extremities (3, 7, 24). Recently,variants of classical Peruvian bartonellosis in which only theverruga phase of the disease was present were observed in thelowland province of Manabi in Ecuador (2). This has led tosuggestions that the milder form of bartonellosis may becaused by less-virulent strains of B. bacilliformis (2).

In the valleys of the Andes where bartonellosis is endemic,approximately 60% of the population are seropositive for thebacterium and 5 to 10% of the population are active carriers ofthe disease (14). Outbreaks of bartonellosis can reach epi-demic proportions in these areas, such as the outbreak of 1870in Oroya, Peru (after which the disease was named), in which

more than 7,000 railroad workers died of the disease. Morerecently, delayed diagnosis resulted in the death of 14 people(88% case fatality) in an epidemic in Peru in 1987 (9). Bar-tonellosis thus remains a significant health problem in regionswhere it is endemic and requires research attention for thedevelopment of rapid tests for diagnosis and treatment of thedisease. Humans are the only known natural reservoir forB. bacilliformis, which suggests that eradication of the diseaseis achievable by vaccinating the population in the regionswhere the disease is endemic.

The skin lesions of the verruga phase of Carrion’s diseaseare very similar to the lesions that are associated with bacillaryangiomatosis (BA), a vascular proliferative disease that ismostly seen among immunocompromised individuals. B. hen-selae, one of the recently included members of the genus Bar-tonella, was identified as a causative agent of BA (21). B. hen-selae has also been implicated in the etiology of cat scratchdisease (CSD) and a number of other disease syndromes.Based on the phylogenetic similarities between B. bacilliformisand B. henselae, it is conceivable that factors common betweenthese two organisms may be responsible for the pathologicalsimilarities between verruga peruana and BA. Identificationand characterization of such factors could lead to a betterunderstanding of the mechanisms of pathogenesis employed bythese organisms.

The present study was initiated to identify and characterizeimmunogenic proteins of B. bacilliformis that are expressedduring the infectious process. We screened a genomic DNAlambda library with serum from a patient who had the chronicverruga phase of bartonellosis and were able to isolate severalimmunoreactive clones expressing bartonella-specific proteins

* Corresponding author. Mailing address: Department of Biology,Georgia State University, Atlanta, GA 30303. Phone: (404) 639-4568.Fax: (404) 639-4436. E-mail: [email protected].

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(18). In this paper we describe the cloning and characterizationof an immunoreactive 43-kDa lipoprotein of B. bacilliformis.

MATERIALS AND METHODS

Bacterial strains, growth conditions, and plasmids. B. bacilliformis strainsKC584 and KC583 were obtained from the American Type Culture Collections(ATCC), Manassas, Va. Both strains were grown on heart infusion agar platessupplemented with 5% defibrinated rabbit blood (BBL-Becton Dickinson,Cockeysville, Md.) at 28°C for 7 to 14 days under humid conditions. B. henselaeHouston-1 (ATCC 49822) strain was grown on the same plates at 32°C in thepresence of 5% CO2 for 5 to 7 days. Bacteria were harvested and resuspended inphosphate-buffered saline (PBS). All E. coli strains were grown at 37°C in mediasupplemented with appropriate antibiotics.

Human sera. The anti-B. bacilliformis human sera used in this study hadindirect fluorescent-antibody assay (IFA) titers ranging from 512 to 1,024. Thesesera were from clinical cases of bartonellosis from Peru. The sera and their titerswere generously provided by Judith Chamberlain of the Department of Preven-tive Medicine and Biometrics, Uniformed Health Services University, Bethesda,Md. The anti-B. henselae human sera used in this study were from suspectedCSD cases and were submitted to the Centers for Disease Control and Preven-tion for confirmative diagnosis. These sera had high titers ($2,048) for B. hen-selae as determined by IFA. Sera with negative IFA titers (#32) to Bartonellaspp. were used as controls.

Preadsorption of sera with E. coli antigens. All of the sera used in this studywere preadsorbed with E. coli antigens to remove cross-reacting antibodies priorto their use in Western blotting. Then, 1.5-ml aliquots of fresh overnight culturesof E. coli strains XL1-Blue MRF9 and JM105 were pelleted by centrifugation.The supernatant was discarded, and the pellet was resuspended in 200 ml ofprotoplasting buffer (15 mM Tris-HCl, pH 8.0; 0.45 M sucrose; 8 mM EDTA).Next, 5 ml of lysozyme (50 mg/ml) was added, and the cells were incubated atroom temperature for 15 min, followed by a 2-min incubation at 37°C. The serawere diluted to a volume of 500 ml and added to the lysed cells. The mixture wasincubated at room temperature for 1 h, with periodic mixing. This was followedby centrifugation for 10 min at 10,000 rpm in a microcentrifuge to remove thecellular debris. The supernatant was carefully collected and used for immuno-assays.

DNA sequencing and analysis. DNA sequencing was done using a model 377automated nucleic acid sequencer (Applied Biosystems, Foster City, Calif.).DNA and protein analyses were performed with the Wisconsin software package(version 8) of the Genetics Computer Group (Madison, Wis.) and DNASTAR(Lasergene, Inc.).

DNA manipulations. The primers that were used for subcloning ORF-401were as follows: forward primer (59-TGA GCA GAA TCC AAT GAG AAGATT CAT GTA-39) and reverse primer (59-ACC TAC CTG CAG TAA ACTGAT ATC ATA GCG-39). The underlined sequences indicate sites for therestriction enzymes EcoRI and PstI for the forward and reverse primers, respec-tively.

The primers were used to amplify the region corresponding to ORF-401 usingpBIPH-1 as the template. The parameters used for the PCR have been describedpreviously (18). The PCR product was purified using Wizard PCR Preps (Pro-mega) and digested with the appropriate restriction enzymes. The digestion ofthe PCR product and the vector pKK223-3 was performed overnight at 37°C.Digestions were stopped by phenol-chloroform extractions, and DNA was pre-cipitated by ethanol precipitation. Insert and vector DNA were gel purified andeluted from the gel by using the freezer-squeeze technique. Briefly, the insert andvector DNA were run on a low-melting-point agarose gel, and the bands were cutout of the gel. Then, 80 ml of TE buffer (10 mM Tris, pH 8.0; 1 mM EDTA) and100 ml of phenol were added to the gel slices, vortexed, and frozen at 270°C.After thawing and centrifugation, the aqueous phase was extracted with a mix-ture of phenol-chloroform-isoamyl alcohol (25:24:1 [vol/vol]), and the DNA wasprecipitated by ethanol precipitation. Ligation of the insert DNA to the vectorwas performed overnight at 16°C. Recombinant clones were analyzed by restric-tion digestion and sequencing of the insert-vector junctions. Expression of pro-teins was studied by Western blotting using polyclonal serum that was generatedagainst the 43-kDa antigen.

PCR-directed mutagenesis. The technique of overlap extension by PCR wasused to mutagenize the polypeptide encoded by ORF-401 (9). Two complemen-tary 30-bp oligonucleotides with the sequences 59-AGGTTCTAGATCTGGCACACAGCGTTTTTT-39 (oligonucleotide A) and 59-AAAAAACGCTGTGTGCCAGATCTAGAACCT-39 (oligonucleotide B) were synthesized for the mutagen-esis. The underlined residues indicate the positions at which these oligonu-cleotides differ from the wild-type sequence to produce a Cys3Ser change atposition 33 of the polypeptide. The nucleotides in boldface denote restrictionenzyme recognition sequences. These oligonucleotides were also designed tointroduce an XbaI restriction site into the resultant PCR product. In the firstround of PCR, oligonucleotides A and B were used in separate reactions alongwith two more oligonucleotides, 59-TGAGCAGAATTCAATGAGAAGATTCATGTA-39 (oligonucleotide C) and 59-ACCTACCTGCAGTAAACTGATATCATAGCG-39 (oligonucleotide D), to generate two overlapping PCR products.These PCR products were purified and used as templates in the second round ofPCR with oligonucleotides C and D to generate a mutated PCR product of 1,300

bp. The mutation was confirmed by sequencing and restriction digestion withXbaI, and the PCR product that contained the mutation was cloned into thevector pKK223-3 to generate the mutant clone pKMUT-9. Expression of themutated polypeptide by pKMUT-9 was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis.

Labeling of lipoproteins with [3H]palmitate. The E. coli strain JM105 carryingpKIP-7, pKMUT-9, or the vector pKK223-3 was grown in Luria broth supple-mented with ampicillin (50 mg/ml). When the optical density at 550 nm reached0.3 to 0.5, 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) and 100 mCi of[9,10(n)-3H]palmitate were added, and the incubation was continued for 2 h. Thecells were pelleted by centrifugation, washed twice in PBS, and resuspended in13 sample buffer in preparation for SDS-PAGE. After electrophoresis, proteinswere fixed by incubating the gel in 5 to 10 volumes of glacial acetic acid-methanol-water (10:20:70) at room temperature with gentle rocking (23). Gelswere impregnated with En3Hance (DuPont NEN), treated with a gel-dryingsolution, dried, and fluorographed overnight at 280°C.

Antibody production. Peptides were dissolved in deionized water at a concen-tration of 2 mg/ml. The polyclonal rabbit antiserum was produced at the animalfacility at Georgia State University. A cocktail consisting of equimolar amountsof each of five peptides was diluted 1:1 with Freund complete adjuvant (Sigma).The diluted peptide cocktail was injected into New Zealand White rabbits (Myr-tle’s Rabbitry, Inc., Thompson Station, Tenn.) for antibody production. Animalswere boosted after 2 weeks with the peptide cocktail mix at a concentration of 1mg/ml in Freund incomplete adjuvant (Sigma). Rabbits were bled after 3 weeks,and the sera were purified by centrifugation. For production of polyclonal rabbitantisera to each of the individual peptides, each of the five peptides was admin-istered to rabbits using a protocol similar to that used for the peptide cocktail.

SDS-PAGE. E. coli strains harboring the recombinant plasmids or vectors wereinduced with 1 mM IPTG prior to SDS-PAGE analysis. Proteins from E. coli andBartonella strains were solubilized in 13 sample buffer (Novex, San Diego, Calif.)at 100°C for 5 min and subjected to electrophoresis on precast 4 to 20% gradientTris-glycine gels (Novex). Gels were run in Tris-glycine SDS-PAGE runningbuffer at 125 V. Separated proteins were either transferred to nitrocellulose,stained with Coomassie brilliant blue, or used for autoradiography as describedabove.

Western blotting. Proteins for immunoblotting were electrophoretically trans-ferred to 0.45-mm (pore size) nitrocellulose membranes (Novex) according to theprotocol of Towbin et al. (27). Transfer was performed in Tris-glycine buffer with20% methanol for 1 h at 100 V with cooling. Membranes were blocked overnightat 4°C in blocking buffer consisting of 5% nonfat milk powder in Tris-bufferedsaline–Tween 20 (20 mM Tris, pH 7.5; 150 mM NaCl; 0.05% Tween 20). Mem-branes were reacted with the primary antibody solutions (in blocking buffer) for2 h at room temperature. The secondary antibody was either goat anti-rabbit oranti-human immunoglobulin G, conjugated to horseradish peroxidase (Kirkeg-aard and Perry Laboratories, Inc., Gaithersburg, Md.) and diluted 1:5,000 inblocking buffer. Membranes were developed with a standard chromogenic sub-strate (TMB Membrane Substrate System; Kirkegaard and Perry).

Nucleotide sequence accession number. The nucleotide sequence of the geneencoding the 43-kDa antigen has been deposited in the GenBnk database andhas been given the accession no. AF157831.

RESULTS

Cloning of the 43-kDa antigen gene. A B. bacilliformis ge-nomic library was constructed using the lambda ZapII systemas described previously (18). The library was screened withserum from a patient from Ecuador who had the chronic ver-ruga phase of bartonellosis. One of the clones (pBIPIM-17)isolated as a result of this immunoscreening expressed a fu-sion protein that was encoded by a partial open reading frameof 741 bp and did not contain a putative ATG start codon(data not shown). To obtain the entire open reading frame,the B. bacilliformis genomic library was rescreened by plaquehybridization using the pBIPIM-17 insert as the probe. Thisscreening resulted in the isolation of three hybridizing clones,pBIPH-1, pBIPH-2, and pBIPH-3, all of which were revealedby DNA sequencing to contain the full-length open readingframe. The deduced 1,206-bp open reading frame was capableof encoding a protein of 401 amino acids (ORF-401) with anestimated molecular mass of approximately 43 kDa (Fig. 1).Examination of the sequence (Fig. 1) revealed a second in-frame ATG codon 9 bp downstream of the first ATG codon(ATG-1) (Fig. 1). This ATG codon is preceded by a Shine-Dalgarno (SD) sequence that is identical to the SD sequenceof E. coli (Fig. 1). Also, based on E. coli SD sequences, the SDpreceding the second ATG is more optimally located. Thus, it

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is possible that the second ATG within the open reading frameis an alternative start site. We have therefore designated thesecond ATG codon as ATG-2 to indicate the possibility that itis used as an alternative site for translational initiation, whichwould result in a protein of 397 amino acids.

The 43-kDa antigen is a homologue of LppB/NlpD. A searchthrough the nucleic acid and protein databases using theBLAST search tool (1) revealed that the predicted amino acidsequence of ORF-401 is homologous to that of a bacteriallipoprotein that was recently identified and designated as novellipoprotein D (NlpD)/lipoprotein B (LppB). Alignment of thededuced amino acid sequence of ORF-401 with some of theknown LppB/NlpD sequences (Fig. 2) revealed that the ho-mology extends throughout the length of the protein and isparticularly striking within a region of approximately 100amino acids near the carboxyl end of the protein (Fig. 2). Thisstrong sequence similarity suggests that the 43-kDa antigen isa homologue of the LppB/NlpD proteins.

ORF-401 expresses a 43-kDa antigen. To verify that ORF-401 codes for a 43-kDa antigen, the open reading frame wassubcloned into pKK223-3 and expressed in Escherichia coli.The resulting recombinant clone, pKIP-7, was found to expressa 43-kDa antigen (Fig. 3, lanes 3 and 4). The recombinantantigen migrated with an antigen of the same size in the celllysates of two strains of B. bacilliformis, KC584 (Fig. 3, lane 5)and KC583 (Fig. 3, lane 6). Lane 2 represents the immuno-reactive fusion protein that is expressed from the recombi-nant vector, pBHIM-17. The 43-kDa band was not present inE. coli cells containing the plasmid vector, pKK223-3 (Fig. 3,lane 1), indicating that the expressed recombinant antigen wasspecific to pKIP-7. The Western blot shown in Fig. 3 wasreacted with a pool of sera from patients with clinical bartonel-losis. The other immunoreactive bands seen in lanes 1 to 4 aremost likely due to the presence of antibodies in the human serathat cross-react with E. coli antigens since they are also presentin E. coli cells containing the plasmid vector (Fig. 3, lane 1).Preadsorption of the sera with E. coli antigens reduced thecross-reactivity but could not eliminate it.

The 43-kDa antigen is a lipoprotein. The possibility that, likethe LppB and NlpD proteins, the 43-kDa antigen is lipid mod-ified was tested by studying the incorporation of [3H]palmiticacid into the protein. E. coli JM105 cells carrying the re-combinant plasmid pKIP-7 were induced with 1 mM IPTGfor expression of the 43-kDa antigen and incubated with[3H]palmitic acid. SDS-PAGE analysis of whole-cell lysates ofpalmitate-labeled E. coli revealed that the 43-kDa antigen isefficiently labeled by [3H]palmitic acid, as is evident from theprominent band migrating at 43 kDa (Fig. 4A, lane 2). Thisband is absent in cell lysates of E. coli harboring the vectorpKK223-3 that were labeled under the same conditions (Fig.4A, lane 1), confirming that it is specific for E. coli expressingthe 43-kDa antigen.

Posttranslational modification of the 43-kDa antigen. Theamino terminus of the polypeptide encoded by ORF-401 con-tains a 32-amino-acid sequence bearing strong homology tosignal peptides found in secreted bacterial proteins (Fig. 2). Atypical bacterial signal sequence consists of three distinct re-gions: a basic region at the N terminus consisting of two to fourbasic amino acids, a core region consisting primarily of hydro-phobic amino acids, and a cleavage region consisting of theconsensus sequence Leu-Ala-Gly-Cys (11, 19). As is evidentfrom Fig. 2, the signal peptide of the 43-kDa antigen has apredominance of positively charged amino acids at the N ter-minus (Arg2-Arg3 and Lys8) and a core that consists primarilyof hydrophobic amino acids. It also contains the sequenceIle-Thr-Gly-Cys, which conforms to the consensus sequenceLeu(Ile)-Ala(Ser/Thr)-Gly(Ala)-Cys that is required for cleav-age of the signal peptide and lipid modification. In bacteriallipoproteins, the cysteine residue is the site at which posttrans-lational lipid modification of the protein occurs, which is fol-lowed by cleavage of the signal peptide (11, 19). To test thepossibility that the 43-kDa antigen was modified at Cys-33 by asimilar mechanism, the cysteine codon (TGT) was changed toa serine codon (TCT) by introducing a single point mutation(G3C) at the appropriate position in pKIP-7. The expressionof the protein from pKMUT-9, the plasmid carrying the mu-tation, was studied by Western blot analysis (Fig. 4B) usingpolyclonal antiserum raised against the 43-kDa antigen (de-scribed in a following section). The Cys3Ser change was ac-companied by a shift in the mobility of the mutant protein, asindicated by the appearance of an immunoreactive proteinband (Fig. 4B, lane 3) that migrates more slowly than the bandthat corresponds to the wild-type protein (lane 2). The changein migration is consistent with the accumulation of a precursor

FIG. 1. Nucleotide and deduced amino acid sequences of ORF-401, express-ing the 43-kDa antigen. Amino acids are shown in single-letter code. An asteriskindicates the stop codon. Translation has been shown from the first in-frameATG codon (ATG-1), which has been highlighted. A second possible transla-tional start site (ATG-2) downstream of ATG-1 has also been indicated byhighlighting. The SD sequences preceding ATG-1 and ATG-2 have been under-lined.

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form of the 43-kDa antigen which has an intact signal peptide.The size of the mutant protein estimated by its rate of migra-tion is approximately 46 kDa, which correlates well with thepredicted increase (3.5 kDa) in the Mr of the protein due to theintact signal peptide.

The lower-molecular-weight immunoreactive bands ob-served in lane 2 of Fig. 4B are most likely products of proteo-lytic degradation of the 43-kDa antigen due to expression athigh levels. The higher-molecular-weight band seen in lane 2most likely represents a small population of unprocessed pro-tein in which the signal peptide is intact, since this band comi-grates with the mutant protein expressed by pKMUT-9 (lane3). This phenomenon may again be related to overexpressionof the 43-kDa antigen. It is possible that when a protein isexpressed at high levels in a “foreign” host (E. coli), some ofthe protein remains unprocessed because of an imbalance be-tween protein synthesis and the availability of signal peptidaseII. However, the majority of the protein is processed, since

there is a much higher proportion of the 43-kDa protein (Fig.4B, lane 2).

Removal of the signal peptidase cleavage site by mutagen-esis should affect the ability of the mutant protein to be post-translationally modified by lipids. To test this assumption,we incubated E. coli cells harboring the mutated plasmid,pKMUT-9, with [3H]palmitic acid under the same conditionsas E. coli cells harboring the wild-type plasmid, pKIP-7. Asseen in Fig. 4A, in contrast to its wild-type counterpart (Fig.4A, lane 2), the mutant protein is unable to incorporate[3H]palmitic acid (Fig. 4A, lane 3). These results suggest thatthe cysteine residue at position 33 of the polypeptide encodedby ORF-401 is the site at which lipid modification occurs,followed by cleavage of the signal peptide.

Immunoreactivity of the 43-kDa antigen with individualsera from patients with clinical bartonellosis. To study thereactivity of the 43-kDa antigen with individual patient sera,sera from five clinical cases of human bartonellosis from Peru

FIG. 2. Alignment of the deduced amino acid sequence of the B. bacilliformis NlpD/LppB homologue with other NlpD/LppB proteins. The alignment wasperformed with the CLUSTAL program, and the shading was done with BOXSHADE. The dark shading indicates identical residues, while the light shading indicatesconserved substitutions. The signal peptide sequence is underlined, and the signal peptidase cleavage site is indicated by the arrowhead. The regions corresponding tosynthetic peptides are numbered and are indicated by discontinuous lines. The RGD sequence is indicated by the heavy underline. Hi, H. influenzae, Hs, H. somnus,Ec, E. coli, Bb, B. bacilliformis. The overall identity and similarity values for the homology of the B. bacilliformis NlpD/LppB protein with the NlpD/LppB homologuesused in the alignment are as follows: H. influenzae, identity, 26.6%, similarity, 42.2%; H. somnus, identity, 19.4%, similarity, 37.6%; E. coli, identity, 24.4%, similarity,34.7%.



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with high IFA titers for B. bacilliformis (512 to 1,024) werereacted with the recombinant 43-kDa antigen expressed inE. coli. The sera were from patients who had either Oroyafever or verruga peruana. As shown in Fig. 5 (lanes 1 to 5), allof the sera tested showed strong reactivity with the 43-kDaantigen. On the other hand, control sera that tested negativefor bartonella as determined by IFA (titer of #32) did not

recognize the 43-kDa antigen (lanes 6 to 10). We studied thecross-reactivity of the 43-kDa antigen with other Bartonellaspecies by Western blot analysis using sera from patients withCSD as confirmed by a high IFA titer for B. henselae (titer of$2,048). All of the five sera that were tested showed strongreactivity to the 43-kDa antigen (lanes 11 to 15), suggestingthat a homologue of the 43-kDa antigen exists in B. henselae.

Generation of polyclonal antiserum against the 43-kDa an-tigen of B. bacilliformis. The antigenic index of the 43-kDaantigen was determined by using the Jameson-Wolf algorithm(DNASTAR). This algorithm uses criteria, such as hydrophi-licity, surface probability, and flexibility, to predict regions ofthe protein that could serve as epitopes involved in generatinga humoral immune response. Five regions of the protein werepredicted as predominant epitopes of the 43-kDa antigen andpeptides corresponding to these regions were synthesized. Thepositions of these peptides are shown in Fig. 2. A mixture ofthe five peptides was used to immunize rabbits for the purposeof generating specific antiserum against the 43-kDa antigen ofB. bacilliformis. The antiserum was found to react very stronglywith a 43-kDa protein in E. coli harboring the recombinantplasmid, pKIP-7, under uninduced (Fig. 6, lane 3) and induced(Fig. 5, lane 4) conditions. This protein band was absent in celllysates of E. coli harboring pKK223-3 (lane 1), indicating thatit is specific to E. coli containing pKIP-7. The rabbit antiserumalso reacted with the fusion protein that was expressed frompBIPIM-17 (Fig. 6, lane 2). Furthermore, the rabbit antiserumwas also able to recognize 43-kDa antigens in both of theknown strains of B. bacilliformis, KC584 and KC583 (lanes 5and 6).

As seen previously in Fig. 4B, the blot revealed additionallower-molecular-weight bands in the lane with E. coli harbor-ing pKIP-7 expressing the 43-kDa antigen under induced con-ditions (Fig. 6, lane 4). As explained in an earlier section, thesebands most likely represent products of proteolytic degrada-tion of the 43-kDa antigen, a phenomenon that is observedwhen the protein is expressed at high (induced) levels but notat basal (uninduced) levels of expression (Fig. 6, lane 3). Thelower-molecular-weight bands are absent in the cell lysates of

FIG. 3. Western immunoblot showing reactivity of the 43-kDa antigen with apool of sera from patients with bartonellosis. Lane 1, E. coli JM105 cells con-taining the plasmid vector, pKK223-3; lane 2, E. coli XL1-Blue cells containingthe recombinant plasmid, pBHIM-17, expressing the truncated fusion protein;lanes 3 and 4, E. coli JM105 cells containing the recombinant plasmid, pKIP-7,expressing the 43-kDa antigen under uninduced (lane 3) and induced (lane 4)conditions; lanes 5 and 6, B. bacilliformis strains KC584 and KC583, respectively.Numbers to the right are approximate molecular masses, in kilodaltons (kDa).The arrow indicates the position of the 43-kDa antigen.

FIG. 4. [3H]palmitate labeling of E. coli cells expressing the 43-kDa antigen. E. coli JM105 cells that contained either pKIP-7 or pKMUT-9 were induced with 1mM IPTG for expression of the wild-type protein (lane 2) or the mutant protein (with uncleaved signal peptide) (lane 3), respectively. Lane 1 represents a control E. coliJM105 strain containing the plasmid vector, pKK223-3. (A) Cells were labeled with [3H]palmitate as described in the text and analyzed by SDS-PAGE andautoradiography. (B) Cells were analyzed by Western immunoblotting using polyclonal rabbit antiserum raised to synthetic peptides of the 43-kDa antigen. Numbersto the left of panel A are approximate molecular masses, in kilodaltons (kDa). The arrow indicates the position of the wild-type, mature form of the 43-kDa antigen.



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B. bacilliformis (lanes 5 and 6), suggesting that this phenome-non is restricted to expression in a foreign host such as E. coli.When pKIP-7 was expressed in a Lon protease-deficient strainof E. coli (BL21), the lower-molecular-weight bands were notdetectable (data not shown), suggesting that Lon is at least oneof the proteases responsible for the degradation phenomenon.Again, as described earlier, the immunoreactive band abovethe 43-kDa band (Fig. 6, lane 4) most likely represents a smallproportion of unprocessed protein containing the intact signalpeptide.

An important observation from the immunoblot is that theserum was highly specific for the 43-kDa antigen of B. bacilli-formis since it did not show reactivity with a protein of thesame size in the B. henselae cell lysate (Fig. 6, lane 7) or inthe cell lysates of other bartonellae such as B. quintana andB. elizabethae (lanes 8 and 9). The immunoblot revealed adiffuse immunoreactive band with a molecular mass of approx-imately 55 kDa in the lanes representing the bartonella celllysates (Fig. 6, lanes 5 to 9). However, this band was observedwhen preimmune sera were reacted with the cell lysates (datanot shown), indicating that it may represent a protein in ourbartonella lysates that cross-reacts with the antibodies presentin the naive rabbit serum.

DISCUSSION

B. bacilliformis, a member of the family Bartonellaceae withinthe alpha-2 subgroup of Proteobacteria, is the etiologic agent ofhuman bartonellosis. Bartonellosis is a unique disease becauseof its biphasic nature, in which bacteria exhibit tropism fordifferent cells in each of the two phases. In the primary acutephase (Oroya fever), the bacteria invade nearly 100% of eryth-rocytes, causing a severe hemolytic anemia. During the sec-ondary chronic phase (verruga peruana), the bartonellae in-vade endothelial cells, which results in wart-like multipletumors involving the skin, mucous membranes, and internalorgans (3, 7). The remarkable difference in disease manifesta-tion during the two stages of bartonellosis suggests a complexinteraction between B. bacilliformis and the human host thatmay involve a multitude of both bacterial and host proteins.

Characterization of antigens expressed during the course of aninfection by B. bacilliformis would be helpful in elucidating thepathogenesis of the disease. Identifying immunogenic antigenswould also be useful for developing tools for the rapid diag-nosis of bartonellosis.

In this study, we have cloned, sequenced, and characterizedan immunogenic 43-kDa antigen of B. bacilliformis. The pre-dicted amino acid sequence of the 43-kDa antigen shows ho-mology to the LppB proteins of Haemophilus somnus and H.influenzae and the NlpD protein of E. coli (Fig. 2). LppB/NlpD

FIG. 5. Western immunoblot of the 43-kDa antigen reacted with individual serum samples from patients with clinical bartonellosis and CSD. Cell lysates of E. coliJM105 cells containing the recombinant plasmid pKIP-7 were electrophoresed on a 4 to 16%, precast, polyacrylamide gel (Novex). Proteins were transferred ontonitrocellulose and reacted with the different sera by using a multiscreen apparatus (Bio-Rad). Sera were diluted 1:200 in 5% Blotto. The rest of the protocol is asdescribed in Materials and Methods. Lanes 1 to 5, serum samples from patients with clinical bartonellosis; lanes 6 to 10, serum samples from patients with negativeIFA titers for Bartonella; lanes 11 to 15, serum samples from patients with CSD. Numbers to the right represent approximate molecular masses, in kilodaltons. Thearrow indicates the position of the 43-kDa antigen.

FIG. 6. Western immunoblot of the 43-kDa antigen reacted with polyclonalrabbit serum raised to a cocktail of synthetic peptides. Lane 1, E. coli JM105 cellscontaining the plasmid vector, pKK223-3; lane 2, E. coli XL1-Blue cells contain-ing the recombinant plasmid, pBHIM-17, expressing the fusion protein; lanes 3and 4, E. coli JM105 cells containing the recombinant plasmid, pKIP-7, express-ing the 43-kDa antigen under uninduced (lane 3) and induced (lane 4) condi-tions; lanes 5 and 6, B. bacilliformis strains KC584 and KC583, respectively; lane7, cell lysate of B. henselae (Houston-1); lanes 8 and 9, cell lysates of B. quintana(Oklahoma) and B. elizabethae (ATCC 49927), respectively. The serum wasdiluted 1:5,000 in 5% Blotto. Numbers to the left are approximate molecularmasses, in kilodaltons (kDa). The arrow indicates the position of the 43-kDaantigen.



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is a recently identified lipoprotein proposed to be located inthe outer membrane of H. somnus, a pathogenic bacteriumthat causes hemophiliosis in cattle (8). In H. somnus, LppB isan immunodominant protein that has been shown to be able tobind the aromatic dye Congo red, a structural analog of heme(26). Congo red binding (Crb1) is a property that has beenused as an indicator of virulence for several pathogenic bacte-ria (5, 13, 20). We have demonstrated that the 43-kDa antigenis able to incorporate [3H]palmitate efficiently, providing ex-perimental evidence that it is indeed a lipoprotein. In addition,mutagenesis of the signal peptidase cleavage site resulted inthe accumulation of a precursor form of the protein that couldnot incorporate [3H]palmitate. These results suggest that the43-kDa antigen of B. bacilliformis is synthesized by a pathwaysimilar to that used by other major lipoproteins in bacteria (11).

We have used Western blotting to demonstrate that serafrom individuals who had classical Peruvian bartonellosis rec-ognize the recombinant 43-kDa antigen that was expressed inE. coli. When reacted with a pool of these sera (Fig. 3), the43-kDa recombinant antigen migrated with an immunoreactiveprotein of the same size in the B. bacilliformis cell lysates,suggesting the presence of the LppB protein in B. bacilliformis.The presence of antibodies against the LppB homologue in thepatient sera indicates that it is an immunogenic protein. Im-munoreactivity of the LppB protein with individual sera frompatients with bartonellosis was also demonstrated (Fig. 5). OurWestern blot analysis data (Fig. 5) also revealed that positiveCSD sera with a high antibody titer to B. henselae recognize the43-kDa antigen of B. bacilliformis. This suggests that a homo-logue of the antigen exists in B. henselae, a bacterium that isphylogenetically closely related to B. bacilliformis. The immu-nogenicity of the 43-kDa antigen in B. bacilliformis and B. hen-selae may have important implications from the perspective ofpathogenesis. Common factors between these two closely re-lated organisms are of special relevance because of the patho-logical similarities between verruga peruana and BA. Futurestudies aimed at testing the ability of the 43-kDa antigen ofB. bacilliformis to bind to endothelial cells may help to identifya possible role in the endothelial cell proliferation that is ahallmark of verruga peruana and BA.

We generated polyclonal antisera against a cocktail of syn-thetic peptides corresponding to antigenic regions within the43-kDa antigen. This serum reacted strongly with the 43-kDaantigen expressed in E. coli and B. bacilliformis but did notcross-react with the E. coli NlpD homologue, as indicated bythe absence of an immunoreactive 43-kDa band in the E. colinegative control that harbored the plasmid (Fig. 6). In addi-tion, the serum did not recognize a 43-kDa protein in theB. henselae cell lysate (Fig. 6), although the presence of theantigen in B. henselae was demonstrated by the reactivity ofthe positive CSD sera with the recombinant 43-kDa antigenexpressed in E. coli (Fig. 5). This suggests that the 43-kDa an-tigens of B. bacilliformis and B. henselae exhibit considerabledivergence, at least within the regions where we synthesizedpeptides, so that they could potentially be useful in the devel-opment of diagnostic tools for differentiating between theseBartonella species.

Investigation of the immunogenicity of the five individualpeptides revealed that only the antiserum against peptide 2(Fig. 2) could recognize the 43-kDa antigen in the B. bacilli-formis cell lysates (data not shown). This suggests that peptide2 is the most immunogenic of the five synthetic peptides and isresponsible for the major part of the immunogenicity of thepeptide cocktail antiserum. It is possible that differences be-tween the B. bacilliformis and B. henselae LppB homologueswithin the region corresponding to peptide 2 could contribute

to the lack of reactivity of the peptide cocktail antiserum withthe B. henselae homologue. The inability of the antiserum torecognize 43-kDa antigens in any of the other bartonellaesuggests that species-specific synthetic peptides based on the43-kDa antigen, such as peptide 2 used in this study, would bepotentially useful for diagnostic purposes. The usefulness ofsynthetic peptides as diagnostic reagents has been demonstrat-ed in previous studies (22).

The presence of the 43-kDa antigen in two strains of B. ba-cilliformis, KC583 and KC584, was demonstrated by immuno-blot analysis using the polyclonal antiserum generated againstthe 43-kDa antigen (Fig. 6). In addition, the 43-kDa antigen isalso present in a putative, uncharacterized Ecuadorian strainof B. bacilliformis (2), as demonstrated by its reactivity withsera from a patient with the milder, atypical form of bartonel-losis. These results suggest that the 43-kDa antigen is a proteinthat is highly conserved among different strains of B. bacilli-formis.

It is possible that, like other NlpD/LppB homologues theLppB protein of B. bacilliformis is exposed at the cell surface.Evidence from fractionation of B. bacilliformis has shown thepresence of antigens of between 42 and 48 kDa, a size rangethat corresponds to that of the LppB protein, in outer mem-brane fractions of B. bacilliformis (14, 16). In addition, exam-ination of the amino acid sequence of the LppB homologue ofB. bacilliformis revealed the presence of a serine residue fol-lowing the cysteine at the signal peptide cleavage site, a featurethat is characteristic of lipoproteins that are exported to theouter membrane (28). Given the importance of cell-surface-exposed factors to pathogenesis, the possible surface localiza-tion of the 43-kDa antigen suggests a potential role in theinfection process of B. bacilliformis. A structural feature thatsupports a role for the 43-kDa antigen in cell adhesion is thepresence of the tripeptide motif, arginine-glycine-aspartate(RGD), near the carboxyl-terminal end of the protein (Fig. 2).The RGD motif, a cell adhesion feature present on bacterialand viral virulence factors, has been proposed to facilitatebinding of pathogens to host cells, promoting their internaliza-tion during the infection process (4, 25). Future studies involv-ing mutagenesis of the RGD sequence of the 43-kDa antigenwill help to determine if this protein plays a role in the inter-action of B. bacilliformis with human endothelial cells whichculminates in verruga peruana.

ACKNOWLEDGMENTS

We thank Russell Regnery at the CDC for giving us the B. bacilli-formis- and B. henselae-specific human sera. We also thank BurtAnderson, currently at the University of South Florida, in whose lab-oratory the initial immunoscreening of the library was done. We aregrateful to P. C. Tai, Department of Biology, GSU, for reviewing themanuscript and for his suggestions regarding expression of the 43-kDaantigen in E. coli. We thank Kelly Bradley for technical assistance withWestern blotting. We also thank the Biotechnology Core Facility at theCDC for synthesis of the oligonucleotides and peptides used in thisstudy.

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Editor: D. L. Burns



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